The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay is used as a major method to evaluate cell viability. However, in some cases, the results may reflect mitochondrial status regardless of viability. Epalrestat (EPS) is currently available for the treatment of diabetic neuropathy. In this study, we report that EPS at near-plasma concentrations increases MTS reduction activity independent of cell number in bovine aortic endothelial cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that plays a pivotal role in inducing the expression of genes encoding detoxifying and defensive proteins. Sulforaphane (an Nrf2 activator) also increased MTS-reducing activity, similar to EPS. Knockdown of Nrf2 by short interfering RNA suppressed EPS-induced MTS reduction. These results suggest that EPS increases MTS reduction activity via the Nrf2 pathway. Furthermore, the results that EPS increases ATP production and that electron transfer chain inhibitors suppress EPS-induced MTS reduction activity suggest that EPS may activate mitochondrial status. Because mitochondrial disorders cause numerous diseases, we suggest that EPS has new beneficial properties that may prevent the development and progression of disorders caused by mitochondrial dysfunction.
References
[1]
Vistica, D.T., Skehan, P., Skudiero, D., Monks, A., Pittman, A. and Boyd, M. (1991) Tetrazolium-Based Assays for Cellular Viability: A Critical Examination of Selected Parameters Affecting Formazan Production. Cancer Research, 51, 2515-2520.
[2]
Alley, M.C., Scudiero, D.A., Monks, A., Hursey, M.L., Czerwinski, M.J., Fine, D.L., Abbott, B.J., Mayo, J.G., Shoemaker, R.H. and Boyd, M.R. (1988) Evaluation of a Soluble Tetrazolium/Formazan Assay for Cell Growth and Drug Sensitivity in Culture Using Human and Other Tumor Cell Lines. Cancer Research, 48, 589-601.
[3]
Goodwin, C.J., Holt, S.J., Downes, S. and Marshall, N.J. (1995) Microculture Tetrazolium Assays: A Comparison between Two New Tetrazolium Salts, XTT and MTS. Journal of Immunol Methods, 179, 95-103.
https://doi.org/10.1016/0022-1759(94)00277-4
[4]
Berg, K., Zhai, L., Chen, M., Kharazmi, A. and Owen, T.C. (1994) The Use of a Water Soluble Formazan Complex to Quantitate the Cell Number and Mitochondrial Function of Leishmania Major Promastigotes. Parasitology Research, 80, 235-239.
https://doi.org/10.1007/bf00932680
[5]
Tominaga, H., Ishiyama, M., Ohseto, F., Sasamoto, K., Hamamoto, T., Suzuki, K. and Watanabe, M. (1999) A Water-Soluble Tetrazolium Salt Useful for Colorimetric Cell Viability Assay. Analytical Communications, 36, 47-50.
https://doi.org/10.1039/A809656B
[6]
Rai, Y., Pathak, R., Kumari, N., Sah, D.K., Pandey, S., Kalra, N., Soni, R., Dwarakanath, B.S. and Bhatt, A.N. (2018) Mitochondrial Biogenesis and Metabolic Hyperactivation Limits the Application of MTT Assay in the Estimation of Radiation Induced Growth Inhibition. Scientific Reports, 8, Article No. 1531.
https://doi.org/10.1038/s41598-018-19930-w
[7]
Javadov, S., Kozlov, A.V. and Camara, A.K.S. (2020) Mitochondria in Health and Diseases. Cells, 9, Article 1177. https://doi.org/10.3390/cells9051177
[8]
Sullivan, L.B. and Chandel, N.S. (2014) Mitochondrial Reactive Oxygen Species and Cancer. Cancer & Metabolism, 2, Article No. 17.
https://doi.org/10.1186/2049-3002-2-17
[9]
Mori, M.P., Penjweini, R., Knutson, J.R., Wang, P.Y. and Hwang, P.M. (2022) Mitochondria and Oxygen Homeostasis. FEBS Journal, 289, 6959-6968.
https://doi.org/10.1111/febs.16115
[10]
Li, X., Fang, P., Mai, J., et al. (2013) Targeting Mitochondrial Reactive Oxygen Species as Novel Therapy for Inflammatory Diseases and Cancers. Journal of Hematology & Oncology, 6, Article No. 19. https://doi.org/10.1186/1756-8722-6-19
[11]
Mikhed, Y., Daiber, A. and Steven, S. (2015) Mitochondrial Oxidative Stress, Mitochondrial DNA Damage and Their Role in Age-Related Vascular Dysfunction. International Journal of Molecular Sciences, 16, 15918-15953.
https://doi.org/10.3390/ijms160715918
[12]
Nowak, W.N., Deng, J., Ruan, X.Z. and Xu, Q. (2017) Reactive Oxygen Species Generation and Atherosclerosis. Arteriosclerosis, Thrombosis, and Vascular Biology, 7, e41-e52. https://doi.org/10.1161/atvbaha.117.309228
[13]
Hayashi, G. and Cortopassi, G. (2015) Oxidative Stress in Inherited Mitochondrial Diseases. Free Radical Biology and Medicine, 88, 10-17.
https://doi.org/10.1016/j.freeradbiomed.2015.05.039
[14]
Ramirez, M.A. and Borja, N.L. (2008) Epalrestat: An Aldose Reductase Inhibitor for the Treatment of Diabetic Neuropathy. Pharmacotherapy, 28, 646-655.
https://doi.org/10.1592/phco.28.5.646
[15]
Ishida, T., In, Y., Inoue, M., Ueno, Y., Tanaka, C. and Hamanaka, N. (1989) Structural Elucidation of Epalrestat(ONO-2235), a Potent Aldose Reductase Inhibitor, and Isomerization of Its Double Bonds. Tetrahedron Letters, 30, 959-962.
https://doi.org/10.1016/S0040-4039(00)95290-0
[16]
Yang, B.B., Hong, Z.W., Zhang, Z., Yu, W., Song, T., Zhu, L.L., Jiang, H.S., Chen, G.T., Chen, Y. and Dai, Y.T. (2019) Epalrestat, an Aldose Reductase Inhibitor, Restores Erectile Function in Streptozocin-Induced Diabetic Rats. International Journal of Impotence Research, 31, 97-104. https://doi.org/10.1089/jop.2016.0103
[17]
Steele, J.W., Faulds, D. and Goa, K.L. (1993) Epalrestat. A Review of Its Pharmacology, and Therapeutic Potential in Late-Onset Complications of Diabetes Mellitus. Drugs & Aging, 3, 532-555. https://doi.org/10.2165/00002512-199303060-00007
[18]
Sato, K., Yama, K., Murao, Y., Tatsunami, R. and Tampo, Y. (2013) Epalrestat Increases Intracellular Glutathione Levels in Schwann Cells through Transcription Regulation. Redox Biology, 2, 15-21. https://doi.org/10.1016/j.redox.2013.11.003
[19]
Yama, K., Sato, K., Abe, N., Murao, Y., Tatsunami, R. and Tampo, Y. (2015) Epalrestat Increases Glutathione, Thioredoxin, and Heme Oxygenase-1 by Stimulating Nrf2 Pathway in Endothelial Cells. Redox Biology, 4, 87-96.
https://doi.org/10.1016/j.redox.2014.12.002
[20]
Connolly, D.T., Knight, M.B., Harakas, N.K., Wittwer, A.J. and Feder, J. (1986) Determination of the Number of Endothelial Cells in Culture Using an Acid Phosphatase Assay. Analytical Biochemistry, 152, 136-140.
https://doi.org/10.1016/0003-2697(86)90131-4
[21]
Kensler, T.W., Wakabayashi, N. and Biswal, S. (2007) Cell Survival Responses to Environmental Stresses via the Keap1-Nrf2-ARE Pathway. Annual Review of Pharmacology and Toxicology, 47, 89-116.
https://doi.org/10.1146/annurev.pharmtox.46.120604.141046
[22]
Kalyanaraman, B. (2013) Teaching the Basics of Redox Biology to Medical and Graduate Students: Oxidants, Antioxidants and Disease Mechanisms. Redox Biology, 1, 244-257. https://doi.org/10.1016/j.redox.2013.01.014
[23]
Kansanen, E., Kuosmanen, M., Leinonen, H. and Levonen, A.L. (2013) The Keap1-Nrf2 Pathway: Mechanisms of Activation and Dysregulation in Cancer. Redox Biology, 1, 45-49. https://doi.org/10.1016/j.redox.2012.10.001
[24]
Collier, A.C. and Pritsos, C.A. (2003) The Mitochondrial Uncoupler Dicumarol Disrupts the MTT Assay. Biochemical Pharmacology, 66, 281-287.
https://doi.org/10.1016/S0006-2952(03)00240-5
[25]
Das, K. and Roychoudhury, A. (2014) Reactive Oxygen Species (ROS) and Response of Antioxidants as ROS-Scavengers during Environmental Stress in Plants. Frontiers in Environmental Science, 2, Article 53.
https://doi.org/10.3389/fenvs.2014.00053
[26]
Kadowaki, T., Kadowaki, H., Mori, Y., et al. (1994) A Subtype of Diabetes Mellitus Associated with a Mutation of Mitochondrial DNA. The New England Journal of Medicine, 330, 962-968. https://doi.org/10.1056/nejm199404073301403
[27]
Murphy, R., Turnbull, D.M., Walker, M. and Hattersleyet, A.T. (2008) Clinical Features, Diagnosis and Management of Maternally Inherited Diabetes and Deafness (MIDD) Associated with the 3243A>G Mitochondrial Point Mutation. Diabetic Medicine, 25, 383-399. https://doi.org/10.1111/j.1464-5491.2008.02359.x
[28]
Birch-Machin, M.A. (2006) The Role of Mitochondria in Ageing and Carcinogenesis. Clinical and Experimental Dermatology, 31, 548-552.
https://doi.org/10.1111/j.1365-2230.2006.02161.x
[29]
Seo, A.Y., Joseph, A.M., Dutta, D., Hwang, J.C., Aris, J.P. and Leeuwenburgh, C. (2010) New Insights into the Role of Mitochondria in Aging: Mitochondrial Dynamics and More. Journal of Cell Science, 123, 2533-2542.
https://doi.org/10.1242/jcs.070490
[30]
Matsuhashi, T., Sato, T., Kanno, S.I., et al. (2017) Mitochonic Acid 5 (MA-5) Facilitates ATP Synthase Oligomerization and Cell Survival in Various Mitochondrial Diseases. EBioMedicine, 20, 27-38. https://doi.org/10.1016/j.ebiom.2017.05.016
[31]
Tonelli, C., Chio, I.I.C. and Tuveson, D.A. (2018) Transcriptional Regulation by Nrf2. Antioxidant & Redox Signaling, 29, 1727-1745.
https://doi.org/10.1089/ars.2017.7342
[32]
Dinkova-Kostova, A.T. and Abramov, A.Y. (2015) The Emerging Role of Nrf2 in Mitochondrial Function. Free Radical Biology and Medicine, 88, 179-188.
https://doi.org/10.1016/j.freeradbiomed.2015.04.036
[33]
Esteras, N., Dinkova-Kostova, A.T. and Abramov, A.Y. (2016) Nrf2 Activation in the Treatment of Neurodegenerative Diseases: A Focus on Its Role in Mitochondrial Bioenergetics and Function. Biological Chemistry, 397, 383-400.
https://doi.org/10.1515/hsz-2015-0295
[34]
Ludtmann, M.H., Angelova, P.R., Zhang, Y., Abramov, A.Y. and Dinkova-Kostova, A.T. (2014) Nrf2 Affects the Efficiency of Mitochondrial Fatty Acid Oxidation. Biochemical Journal, 457, 415-424. https://doi.org/10.1042/bj20130863
[35]
Holmstrom, K.M., Kostov, R.V. and Dinkova-Kostova, A.T. (2016) The Multifaceted Role of Nrf2 in Mitochondrial Function. Current Opinion Toxicology, 1, 80-91. https://doi.org/10.1016/j.cotox.2016.10.002
[36]
Dinkova-Kostova, A.T., Fahey, J.W., Kostov, R.V. and Kensler, T.W. (2017) KEAP1 and Done? Targeting the NRF2 Pathway with Sulforaphane. Trends in Food Science & Technology, 69, 257-269. https://doi.org/10.1016/j.tifs.2017.02.002
[37]
Pan, J., Shen, F., Tian, K., Wang, M., Xi, Y., Li, J. and Huang, Z. (2019) Triptolide Induces Oxidative Damage in NRK-52E Cells through Facilitating Nrf2 Degradation by Ubiquitination via the GSK-3β/Fyn Pathway. Toxicology in Vitro. 58, 187-194. https://doi.org/10.1016/j.tiv.2019.03.032
[38]
Jardim, F.R., Almeida, F.J.S., Luckachaki, M.D. and Oliveira, M.R. (2020) Effects of Sulforaphane on Brain Mitochondria: Mechanistic View and Future Directions. Journal of Zhejiang University Science B, 21, 263-279.
https://doi.org/10.1631/jzus.b1900614
[39]
Carrasco-Pozo, C., Tan, K.N., Gotteland, M. and Borges, K. (2017) Sulforaphane Protects against High Cholesterol-Induced Mitochondrial Bioenergetics Impairments, Inflammation, and Oxidative Stress and Preserves Pancreatic β-Cells Function. Oxidative Medicine and Cell Longevity, 2017, Article ID: 3839756.
https://doi.org/10.1155/2017/3839756
[40]
Qiu, L., Luo, Y. and Chen, X. (2018) Quercetin Attenuates Mitochondrial Dysfunction and Biogenesis via Upregulated AMPK/SIRT1 Signaling Pathway in OA Rats. Biomedicine & Pharmacotherapy, 103, 1585-1591.
https://doi.org/10.1016/j.biopha.2018.05.003
[41]
Li, X., Wang, H., Gao, Y., Li, L., Tang, C., Wen, G., Zhou, Y., Zhou, M., Mao, L. and Fan, Y. (2016) Protective Effects of Quercetin on Mitochondrial Biogenesis in Experimental Traumatic Brain Injury via the Nrf2 Signaling Pathway. PLOS ONE, 11, e0164237. https://doi.org/10.1371/journal.pone.0164237
[42]
Zhao, R.Z., Jiang, S., Zhang, L. and Yu, Z.B. (2019) Mitochondrial Electron Transport Chain, ROS Generation and Uncoupling (Review). International Journal of Molecular Medicine, 44, 3-15. https://doi.org/10.3892/ijmm.2019.4188
[43]
Sahoo, B.M., Ravi, Kumar, B.V.V., Sruti, J., Mahapatra, M.K., Banik, B.K. and Borah, P. (2021) Drug Repurposing Strategy (DRS): Emerging Approach to Identify Potential Therapeutics for Treatment of Novel Coronavirus Infection. Frontiers Molecular Biosciences, 8, Article 628144.
https://doi.org/10.3389/fmolb.2021.628144
[44]
Hotta, N., Akanuma, Y., Kawamori, R., Matsuoka, K., Oka, Y., Shichiri, M., Toyota, T., Nakashima, M., Yoshimura, I., Sakamoto, N. and Shigeta, Y. (2006) Long-Term Clinical Effects of Epalrestat, an Aldose Reductase Inhibitor, on Diabetic Peripheral Neuropathy: The 3-Year, Multicenter, Comparative Aldose Reductase Inhibitor-Diabetes Complications Trial. Diabetes Care, 29, 1538-1544.
https://doi.org/10.2337/dc05-2370
